Diffuse Pharmacophoric Domains of Vasoactive Intestinal Peptide (VIP) and Further Insights into the Interaction of VIP with the N-terminal Ectodomain of Human VPAC1 Receptor by Photoaffinity Labeling with [Bpa6]-VIP

Abstract

The neuropeptide vasoactive intestinal peptide (VIP) 1 is present in both central and peripheral nervous systems as well as in immune cells (1). It controls a large array of biological functions in the brain and peripheral organs (1) and was shown recently to exert potent anti-inflammatory actions (2). The two cloned VIP receptors also bind with high affinity another neuropeptide, the pituitary adenylyl cyclase-activating peptide, and have been named VPAC 2 receptors thereby (3). They are class II G protein-coupled receptor-like receptors for all peptides structurally related to VIP and also receptors for parathyroid hormone, calcitonin, and corticotropin-releasing factor (3).The VPAC1 receptor is prototypic of class II G protein-coupled receptors and has been extensively studied by molecular biology techniques including site-directed mutagenesis and molecular chimerism (for review see Ref.3). These studies made it possible to delineate the receptor domains involved in high affinity VIP binding (3), selectivity toward some natural peptide agonists (4, 5) and also activation of adenylyl cyclase (6). With respect to VIP binding, it appeared that the N-terminal ectodomain of the receptor plays a crucial role, although it is not sufficient to ensure high affinity (3). A three-dimensional model of the N-terminal ectodomain of the hVPAC1 receptor has been developed suggesting the existence of a VIP-binding groove within this domain (7). Despite these extensive studies of the structure-function relationship of hVPAC1 receptor, the physical sites of interaction between VIP and its receptors had remained elusive until recent photoaffinity showing labeling experiments that the side chain of position 22 of VIP is in direct contact with one edge of the putative binding groove in the N-terminal ectodomain (8).It is well known that VIP has diffuse pharmacophoric domains, with the amino acid residues important for biological activity being distributed along the whole 28-amino acid peptide chain (9). In this context, we further explored the contact sites between VIP and the hVPAC1 receptor by incorporating a photoactivable benzophenone group on the side chain at position 6 of VIP. This was done by substituting para-benzoyl-L-Phe (Bpa) for phenylalanine 6. This site of incorporation was selected for several reasons: (i) Phe 6 is important for the biological activity of VIP (9) and is strictly conserved in all natural hormones structurally related to VIP (1). (ii) The substitution of Bpa for phenylalanine keeps an aromatic residue, and we expected that, even though Phe 6 is important for biological activity, the [Bpa 6 ]-VIP probe should keep reasonable affinity for the receptor. (iii) Position 6 and the previously explored position 22 (8) are at the two ends of the central ␣-helical domain of VIP (9, 10). We report here that the amino acid in

Peptide Agonist Docking in the N-terminal Ectodomain of a Class II G Protein-coupled Receptor, the VPAC1 Receptor

“…Radioreceptorassay and Adenylyl Cyclase Activity Measurement-Ligand binding to membrane preparations was carried out as described (12) (10,11). After a 1-h incubation, cells were washed and pelleted by centrifugation, and the pellets were resuspended in 4 ml of 20 mM HEPES buffer containing 1 mM phenylmethylsulfonyl fluoride and 0.1 mM tosyl-L-lysine chloromethyl ketone.…”

“…Photoaffinity-labeled Receptor Cleavage-Cleavage of labeled hVPAC1 receptor or mutants with CNBr, peptide N-glycosidase F, or endoproteinase Glu-C was performed as described (10,11). Products of cleavage were resolved on NuPAGE 4 -12% Bis-Tris gel using MES SDS running buffer system from Invitrogen under reducing conditions in the presence of 20 mM dithiothreitol (10,11).…”

“…Products of cleavage were resolved on NuPAGE 4 -12% Bis-Tris gel using MES SDS running buffer system from Invitrogen under reducing conditions in the presence of 20 mM dithiothreitol (10,11). After electrophoresis, gels were dried and exposed to x-ray film as described (10).…”

“…It appeared that the N-ted of the VPAC1 receptor plays a crucial role for VIP binding, although it is not sufficient to ensure high affinity (9). The physical sites of interaction between VIP and its receptor had remained elusive until recent photoaffinity experiments showing that the side chains of positions 6 and 22 of VIP are in direct contact with the N-ted of the receptor (10,11). Since it is well known that VIP has diffuse pharmacophoric domains, with the amino acid residues important for biological activity being distributed along the whole 28-amino acid peptide chain (12), incorporation of a photoactivable group on the side chain at other positions of VIP is clearly needed.…”

Identification of Ligand Binding Site of Phytosulfokine Receptor by On-column Photoaffinity Labeling

“…These difficulties are further compounded by low concentrations of binding proteins, especially in the case of transmembrane receptors and channels that cannot be functionally overexpressed in bacteria. In fact, most known cross-linked regions of membrane-localized proteins have been identified by analyzing chemical and enzymatic fragmentation patterns of labeled proteins using radioactive photoaffinity ligands rather than direct MS analysis of purified peptide fragments cross-linked with photoaffinity ligands (12)(13)(14).…”